ACTH stimulates adrenal steroidogenesis at a site between cholesterol and pregnenolone, a series of reactions which take place in adrenal mitochondria. These reactions are inhibited by the end-product, pregnenolone, at the transformation of cholesterol to 20 alpha- hydroxycholesterol. Consequently, the characteristics of pregnenolone synthesis in adrenal mitochondria from control and ACTH stimulated animals and the effects of pregnenolone on this transformation are under investigation. A rapidly turning-over protein has been implicated in the action of ACTH. On the basis that phosphorylation of this protein via a cyclic- AMP dependent protein kinase is involved in ACTH action an ACTH-induced increase in a phosphorylated protein is being sought. Hydroxylation of steroids in the 11-position also takes place in adrenal mitochondria. Characteristics of this reaction and its relationship to pregnenolone formation are being studied.